Protective effect of Allium sativum (garlic) aqueous extract against lead-induced oxidative stress in the rat brain, liver, and kidney.

The present investigation was undertaken to evaluate the ameliorative activity of Allium sativum against lead-induced oxidative stress in the brain, liver, and kidney of male rats. Four groups of male Wistar strain rats (100-120 g) were taken: group 1 received 1000 mg/L sodium acetate and group 2 was given 1000 mg/L lead acetate through drinking water for 2 weeks. Group 3 and 4 were treated with 250 mg/kg body weight/day of A. sativum and 500 mg/kg body weight/day of A. sativum, respectively, by oral intubation for a period of 2 weeks along with lead acetate. The rats were sacrificed after treatment and the brain, liver, and kidney were isolated on ice. In the brain, four important regions namely the hippocampus, cerebellum, cerebral cortex, and brain stem were separated and used for the present investigation. Blood was also drawn by cardiac puncture and preserved in heparinized vials at 4 °C for estimation of delta-aminolevulinic acid dehydratase (ALAD) activity. The results showed a significant (p < 0.05) increase in reactive oxygen species (ROS), lipid peroxidation products (LPP), total protein carbonyl content (TPCC), and lead in the selected brain regions, liver, and kidney of lead-exposed group compared with their respective controls. Blood delta-ALAD activity showed a significant (p < 0.05) decrease in the lead-exposed rats. However, the concomitant administration of A. sativum resulted in tissue-specific recovery of oxidative stress parameters namely ROS, LPP, and TPCC. A. sativum treatment also restored the blood delta-ALAD activity back to control. Overall, our results indicate that A. sativum administration could be an effective antioxidant treatment strategy for lead-induced oxidative insult.

Lead induced alterations in nitrite and nitrate levels in different regions of the rat brain.

Nitric oxide (NO) is a free radical synthesized by nitric oxide synthase (NOS) during the conversion of L-arginine to citrulline. Lead (Pb) affects neuronal functioning in the rat brain. Nitric oxide, a neuronal messenger has a short half life and converts immediately into nitrite and nitrate. The present study is designed to determine lead-induced alterations in NO production by measuring nitrite and nitrate in the cerebellum, the hippocampus, the frontal cortex and the brain stem of the rat brain. Male Sprague-Dawley rats were treated with lead acetate (5 and 15 mg/kg body wt.) by intraperitoneal injection. The control and experimental rats were sacrificed at the end of 7 and 14 days after treatment and different regions of the brain were isolated. Nitrite and nitrate (NOx) levels were estimated by the chemiluminescent method using the NOA 280 (Sievers). The data suggested dose-dependent and region-specific responses to lead. Both treatments of lead reduced NOx levels in the cerebellum and the hippocampus. However, the frontal cortex and the brain stem responded differently to Pb exposure. NOx levels in the frontal cortex were significantly increased in rats treated with low and high doses of Pb for 7 days but not in rats treated for 14 days, whereas in the brain stem, NOx levels were increased in a dose- and time-dependent manner. Although, the response was time-dependent, the variation between 7- and 14-day treatment was not clearly delineated. These results provide additional evidence that Pb exposure alters NO-production in rat brain leading to neuronal dysfunction.

In vitro metal inhibition of N-methyl-D-aspartate specific glutamate receptor binding in neonatal and adult rat brain.

The in vitro effect of methyl mercury (MM) and lead (Pb) on N-methyl-D-aspartate (NMDA)-specific glutamate receptor binding in neonatal (10 days old) and adult rat brain was investigated. The cerebral cortex was isolated from the neonatal and adult male Sprague-Dawley rats and the synaptic plasma membranes were prepared to study the NMDA-specific glutamate receptor binding by using (3H)-glutamic acid. The metal salts such as methyl mercury chloride and lead acetate were used to study the effect of MM and Pb. Both MM and Pb significantly inhibited the receptor binding in neonatal and adult rat brain in a concentration-dependent manner. MM (IC50:0.95 +/- 0.08 microM) was more potent in inhibiting the receptor binding than Pb (IC50:60 +/- 7 microM) in neonatal rat brain. A similar high potency was observed for MM than Pb in adult rat brain but the IC50 values are very high (70 +/- 6 microM and 300 +/- 24 microM respectively) indicating less effect compared to neonatal brain. The data suggest that NMDA-receptor binding was more sensitive to MM and Pb in neonatal brain than in adult. MM was more effective than Pb because of its more lipophilicity.

Interaction of metals with muscarinic cholinoceptor and adrenoceptor binding, and agonist-stimulated inositol phospholipid hydrolysis in rat brain.

In vitro mercury (Hg) or lead (Pb) effectively inhibited the binding of 3H-quinuclidinyl-benzilate (QNB) (a muscarinic cholinoceptor antagonist) and 3H-prazosin (an alpha 1-adrenoceptor antagonist) to their receptors in cerebellar and cerebral cortex membranes in a concentration-dependent manner. Hg was more potent than Pb. When the rats were treated with Hg (5 mg/kg body wt) or Pb (25 mg/kg body wt) for 24 hr, a decrease in 3H-prazosin and an increase in 3H-QNB receptor binding were observed in cerebral cortex. There was no alteration in 3H-prazosin binding in cerebellum with the above treatment of metals, but 3H-QNB binding in cerebellum was significantly inhibited by Hg. However, both 3H-prazosin and 3H-QNB receptor bindings were significantly decreased in cerebellum of rats treated for 7 days with Hg (1 mg/kg body wt/day) or Pb (25 mg/ kg body wt/day). But in cerebral cortex of rats treated with these metals for 7 days, a decrease in 3H-prazosin and an increase in 3H-QNB receptor binding activities were noticed. There was a significant decrease in phospholipid content in cerebral cortex but not in cerebellum of rats treated with these metals for 7 days. At 100 microM concentration carbachol or acetylcholine or norepinephrine stimulated 3H-inositol incorporation and 3H-inositol phosphate (IP) formation in rat cerebral cortical slices. Hg or Pb in vitro though increased the agonist-stimulated 3H-inositol incorporation, 3H-IP formation was not significantly altered. The present investigation demonstrates the differential responses by alpha 1-adrenoceptor and muscarinic cholinoceptor in cerebellum and cerebral cortex of rat to in vitro and in vivo effects of Hg or Pb.

In vitro and in vivo effects of lead, methyl mercury and mercury on inositol 1,4,5-trisphosphate and 1,3,4,5-tetrakisphosphate receptor bindings in rat brain.

In vitro and in vivo effects of mercury (Hg), methyl mercury (MM) and lead (Pb) on [3H]inositol 1,4,5-trisphosphate (IP3) and [3H]inositol 1,3,4,5-tetrakisphosphate (IP4) receptor binding in the Sprague-Dawley rat brain cerebellar membranes were studied. In vitro studies indicate that binding of [3H]IP3 and [3H]IP4 to cerebellar membranes was inhibited by Hg while they were stimulated by MM or Pb in a concentration-dependent manner. MM was more potent (EC50 3.4 microM) than Pb (EC50 18.2 microM) in stimulating the [3H]IP3 receptor binding activity whereas Pb (IC50 30 microM) was more potent than MM (IC50 133 microM) in stimulating the [3H]IP4 receptor binding. When the rats were treated (i.p) with Hg (5 mg/kg body wt.) or MM (5 mg/kg body wt.) or Pb (25 mg/kg body wt.) for 3 or 24 h, no significant alterations in [3H]IP3 receptor binding were observed in cerebellum and cerebral cortex. But the above treatment of Pb or MM for 3 or 24 h to rats resulted in an increase of [3H]IP4 receptor binding in the membranes of cerebral cortex. However, the rats treated with Hg (1 mg/kg body wt./day) or Pb (25 mg/kg body wt./day) for 7 days did not show any alteration in binding of [3H]IP3 to its receptors in cerebellar membranes but an increase in this receptor binding was noticed with the treatment of MM (2.5 mg/kg body wt./day) for 7 days. The cerebellum and cerebral cortex of rats with the above treatment of MM or Pb for 7 days exhibited an increase in [3H]IP4 receptor binding. These in vitro and in vivo data suggest that alterations in inositol polyphosphate receptor binding by metals could result in alterations in intracellular calcium levels which may influence neuronal activity.

Inhibition of calcium transport by mercury salts in rat cerebellum and cerebral cortex in vitro.

The present investigation was initiated to study the differential effects of mercury salts on calcium pump activity of rat cerebellum and cerebral cortex in vitro. The calcium pump activity was studied by assaying calcium-adenosine triphosphatase (Ca(2+)-ATPase) in synaptic plasma membranes (SPMs) and microsomes of cerebellum and cerebral cortex in the presence of different micromolar concentrations of mercury and methylmercury. The 45Ca uptake in microsomes of cerebellum and cerebral cortex was also determined in the presence of both the salts of mercury. The SPMs and microsomes were prepared by differential centrifugation using a sucrose gradient (0.8/1.2 M). The Ca(2+)-ATPase activity was determined by estimating the inorganic phosphate. The 45Ca uptake was measured in microsomes by using 45CaCl2. Calcium-ATPase in SPMs was significantly inhibited by these two mercury salts in a concentration-dependent manner. In cerebellum and cortex, the IC50 values for mercuric chloride were 0.065 and 0.081 microM, respectively, whereas they were 0.354 and 0.384 microM for methylmercury chloride, indicating that mercuric chloride was more potent in inhibiting the plasma membrane Ca2+ extrusion process when compared to methylmercury chloride. As seen in SPMs, Ca(2+)-ATPase and 45Ca uptake in microsomes were also significantly inhibited in both cerebellum and cortex by mercury salts in a concentration-dependent manner, the effect being greater with mercuric chloride. These results indicate that both mercury and methylmercury inhibited the Ca2+ pumps located in SPMs and microsomes differentially, and to some extent the effects were also region specific.

In vitro effect of lead on Ca(2+)-ATPase in synaptic plasma membranes and microsomes of rat cerebral cortex and cerebellum.

Earlier investigations from this laboratory suggest that lead interferes with the calcium homeostasis of rat brain through modulation of an inositol polyphosphate second-messenger system. The present investigation was initiated to study the comparative effects of lead chloride and lead acetate on synaptosomal and microsomal Ca(2+)-ATPase of rat cerebral cortex and cerebellum in vitro. The synaptic plasma membranes and microsomes were prepared by using sucrose gradient (1.2-0.8 M). The assay of Ca(2+)-ATPase was done by hydrolysis of ATP and the liberated inorganic phosphate was estimated. Both lead chloride and lead acetate at micromolar concentrations significantly inhibited the Ca(2+)-ATPase of synaptic plasma membranes and microsomes of cerebral cortex and cerebellum in a concentration-dependent manner. The IC50 values of Ca(2+)-ATPase for both lead salts in synaptosomes were significantly lower (P < 0.05) than that of microsomes, indicating more sensitivity. Significantly (P < 0.05) lower IC50 values for both synaptosomal and microsomal Ca(2+)-ATPase were obtained for lead acetate than for lead chloride. The results suggest that lead acetate is more potent than lead chloride in inhibiting the Ca(2+)-ATPase. The microsomal Ca2+ uptake was also studied in cerebellum and cerebral cortex in the presence of different concentrations of both the lead salts. However, these lead salts in vitro did not reveal a significant (P < 0.05) change in the microsomal Ca2+ uptake of cerebellum and cerebral cortex. But earlier investigations indicated that in vitro lead (0.25-2 microM) inhibits inositol 1,4,5-triphosphate-mediated Ca2+ uptake and release in microsomes of rat cerebellum.

Modulation of protein kinase C by heavy metals.

Protein kinase C (PKC) regulates a variety of intracellular and extracellular signals across the neuronal membrane. PKC requires calcium and phospholipid, particularly phosphatidylserine (PS) for its activation. The data indicates that mercury (Hg), lead (Pb) and methyl mercury (CH3Hg) in vitro inhibited the PKC activity at micromolar concentrations in a concentration-dependent manner with IC50 values of 1.5, 2.12 and 0.22 microM, respectively. The IC50 values indicate that CH3Hg was more potent in inhibiting the enzyme activity than Hg or Pb. The basal PKC activity was also inhibited by Pb or Hg. However, the PS-stimulated PKC activity was more sensitive to Pb or Hg than the basal enzyme. The phorbol ester binding to PKC was also found to be inhibited by micromolar concentrations of these metals in vitro. Hg and CH3Hg were more potent inhibitors of phorbol ester binding than Pb. Dithiothreitol (DTT), a dithiol, but not glutathione (GSH) a monothiol, protected the activities of both PS-stimulated and basal PKC from metal-inhibition in a concentration-dependent manner. The present study suggests that the dithiols but not monothiols effectively protect metal-inhibited activity of PKC in rat brain.

In vitro interaction of heavy metals with ouabain receptors in rat brain microsomes.

This study investigates the influence of heavy metals on ouabain-binding in presence of thiol (sulfhydryl) compounds. The data on in vitro effects of mercury (Hg), lead (Pb) and cadmium (Cd) showed significant inhibition of 3H-ouabain binding to microsomal membrane in a concentration-dependent manner. Maximum inhibition of 3H-ouabain binding was observed at 1 microM for Hg and 100 microM each for Pb and Cd. Preincubation with monothiol (L-cysteine or glutathione) or dithiol (dithiothreitol) protected inhibition of 3H-ouabain binding to the membranes by Hg or Pb. Dithiol but not monothiols partially protected Cd-inhibition. The present data confirm that the heavy metals interact with ouabain receptors in a manner similar to SH-blocking agents and protection of metal-inhibited 3H-ouabain binding by thiol compounds is metal specific.

Effects of lead on pH and temperature-dependent substrate-activation kinetics of ATPase system and its protection by thiol compounds in rat brain.

Lead (Pb) inhibited the activities of Na(+)-K+ ATPase (IC50 = 2.0 x 10(-6) M), K(+)-Para-Nitrophenyl phosphatase (PNPPase) (IC50 = 3.5 x 10(-6) M) and [3H]-ouabain binding (IC50 = 4.0 x 10(-5) M) in rat brain P2 fraction. A variable temperature or pH significantly elevated the inhibition of Na(+)-K+ ATPase by Pb in buffered acidic, neutral and alkaline pH ranges. Noncompetitive inhibition with respect to activation of Na(+)-K+ ATPase by ATP was indicated by a variation in Vmax values with no significant changes in Km values at any temperature studied. In the presence of Pb, for Na(+)-K+ ATPase at pH 6.5 and 8.5, Vmax was decreased with an increase in Km values suggesting a mixed type of inhibition. Sulfhydryl agents such as dithiothreitol (DTT) and cysteine (Cyst), but not glutathione (GSH) offered varied levels of protection against Pb-inhibition of Na(+)-K+ ATPase at pH 7.5 and 8.5. The present data suggest that inhibition of Na(+)-K+ ATPase by Pb is both temperature and pH-dependent. These results also indicate that Pb inhibited Na(+)-K+ ATPase by interfering with phosphorylation of enzyme molecule and dephosphorylation of the enzyme-phosphoryl complex and exerted an effect similar to that of SH-blocking agents.

Effect of mercuric chloride on the kinetics of cationic and substrate activation of the rat brain microsomal ATPase system.

Mercuric chloride (HgCl2), a neurotoxic compound, inhibited the adenosine triphosphatase (ATPase) system in a concentration-dependent manner. Hydrolysis of ATP was linear with time with or without HgCl2 in the reaction mixtures. Higher inhibition of (Na(+)-K+)ATPase activity by HgCl2 was observed in alkaline (8.0 to 9.0) pH and at lower temperatures (17 to 32 degrees). Activation energy values were increased slightly in the presence of HgCl2. Activation of (Na(+)-K+)ATPase by ATP in the presence of HgCl2 showed a decrease in Vmax from 15.29 to 5.0 mumol of inorganic phosphate (Pi)/mg protein/hr with no change in Km. Similarly, activation of K(+)-stimulated p-nitrophenyl phosphatase (K(+)-PNPPase) in the presence of HgCl2 showed a decrease in Vmax from 3.26 to 1.35 mumols of p-nitrophenol (PNP)/mg protein/hr with no change in Km. K(+)-activation kinetic studies indicated that HgCl2 decreased Vmax from 14.01 to 4.30 mumols Pi/mg protein/hr in the case of (Na(+)-K+)ATPase and from 3.45 to 2.40 mumols PNP/mg protein/hr in the case of K(+)-PNPPase with no changes in Km. Na(+)-activation of (Na(+)-K+)ATPase in the presence of HgCl2 showed a decrease in Vmax from 11.06 to 3.23 mumols Pi/mg protein/hr and an increase in Km from 1.06 to 2.08 mM. Preincubation of microsomes with sulfhydryl (SH) agents dithiothreitol, cysteine and glutathione protected HgCl2-inhibition of (Na(+)-K+)ATPase. The data suggest that HgCl2 inhibited (Na(+)-K+)ATPase by interfering with the dephosphorylation of the enzyme-phosphoryl complex.

Inhibition of rat brain microsomal Na+/K(+)-ATPase and ouabain binding by mercuric chloride.

This study concerned the effects of mercuric chloride on Na+/K(+)-ATPase and [3H]ouabain binding in rat brain microsomes in vitro. The data showed that HgCl2 inhibited Na+/K(+)-ATPase effectively at micromolar concentrations. The degree of inhibition was decreased with increases in enzyme concentration and incubation time. Variations in the ionic strength of Na+ and K+ did not alter the percent inhibition of Na+/K(+)-ATPase activity by HgCl2. Repeated washings partially restored enzyme activity. The binding of [3H]ouabain to microsomal membranes was inhibited by HgCl2 in a concentration-dependent manner. Cumulative inhibition studies with HgCl2 and ouabain indicated that these inhibitors did not act concurrently and independently on Na+/K(+)-ATPase.

Effects of lead on K(+)-para-nitrophenyl phosphatase activity and protection by thiol reagents.

Lead (Pb) inhibited K(+)-stimulated para-nitrophenyl phosphatase (K(+)-PNPPase) of rat brain P2 fraction in a concentration-dependent manner with IC50 3.5 microM. Altered pH versus activity demonstrated comparable inhibitions by Pb in buffered acidic, neutral and alkaline pH ranges. Inhibition of enzyme activity was higher at lower temperatures (17-27 degrees C) compared to 37 degrees C. Preincubation of enzyme with sulfhydryl (-SH) agents such as cysteine (Cyst) and dithiothreitol (DTT) but not glutathione (GSH) protected against Pb-inhibition. Uncompetitive type of inhibition with respect to the activation of K+ was indicated by a decrease in Vmax from 16.2 to 8.37 mumoles of para-nitrophenol (PNP)/mg protein/hr and Km from 18.99 to 12.39 mM. Kinetic studies on substrate (p-nitrophenyl phosphate) activation in the presence of Pb (3.5 microM) indicated a significant decrease in Vmax from 8.94 to 4.69 mumoles of PNP/mg protein/hr with no change in Km. Cyst (3 microM) and DTT (10 microM) reversed the Pb-inhibited Vmax from 4.69 to 8.38 and 7.24 mumoles of PNP/mg protein/hr respectively. These results suggest that the critical conformational property of K(+)-PNPPase is sensitive to Pb. The data also indicates that the Pb inhibits Na(+)-K+ ATPase system by interacting with dephosphorylation of the enzyme-phosphoryl complex, while Cyst and DTT protected against Pb-inhibition.